Effects of copper and florfenicol on nirS- and nirK-type denitrifier communities and related antibiotic resistance in vegetable soils.

Denitrification play an important role in nitrogen cycle and is affected by veterinary drugs entering agricultural soils. In the present study, the effects of copper and florfenicol on denitrification, related antibiotic resistance and environmental variables were characterized using real-time quantitative PCR (qPCR) and amplicon sequencing in a short-term (30 d) soil model experiment. Drug additions significantly decreased the nirS gene abundance (P < 0.05) but maximized the abundance of gene nirK in soil containing florfenicol and moderate copper levels (150 mg kg-1). Surprisingly, copper additions decreased the fexB gene abundance, however, the abundance of gene pcoD significantly increased in soils containing florfenicol, moderate copper levels (150 mg kg-1), and florfenicol and low copper levels (30 mg kg-1), respectively (P < 0.05). Overall, the nirK-type community composition was more complex than that of nirS-type but Proteobacteria predominated (> 90%) in both communities. Correlation analysis indicated that the gene abundance of fexB was highly correlated with NH4+-N (P < 0.05) and NO3--N (P < -0.01), and floR gene abundance was positively correlated with nirK (P < 0.01). Besides, the abundance of nirS-type genera Bradyrhizobium and Pseudomonas were obviously related to total organic matter (TOM), total nitrogen (TN) or total phosphorus (TP) (P < 0.05), while the abundance of nirK-type Rhizobium, Sphingomonas and Bosea showed a significantly correlated with TOM, TN or copper contents (P < 0.05). Taken together, copper and florfenicol contamination increased the possibility of durg resistance genes spread in agricultural soils through nitrogen transformation.

Effects of sulfamethoxazole on nitrogen removal and molecular ecological network in integrated vertical-flow constructed wetland.

Response of nitrogen removal efficiency and microbial interactions to organic pollution has been a major issue in wastewater treatment system. However, the nitrogen removal efficiency and interactions among microbial community under antibiotics press is still unclear. Thus, the effect of sulfamethoxazole (SMX) on nitrogen removal and microbial responses of IVCWs was investigated through recorded the nitrogen removal efficiency before and after adding SMX and random matrix theory (RMT)-based network analysis. Results showed that better NH4+-N removal (>90%) after a long period of operation were achieved in IVCWs, but NO3--N was accumulated. However, nitrate removal rates were significantly increased after long-term exposure (60 d) to 100 µgL-1 SMX (from 27.35% to 35.57%) with relatively high SMX removal (53.50%). Surprisingly, the ammonia nitrogen removal rate (90.07-92.70%) were not significantly affected by SMX in IVCWs. Moreover, the bacterial richness was decreased and the bacterial community structures were altered by the presence of SMX, especially those of nitrogen-transforming microorganisms. Molecular ecological network analysis suggested that SMX had positive influences on denitrifying bacteria interactions but reduced the network complexity and microbial interactions on whole molecular network, and among-module connections were weakened obviously at SMX.

Response characteristics of nirS-type denitrifier Paracoccus denitrificans under florfenicol stress.

Florfenicol (FF) is widely used in aquaculture and can interfere with denitrification when released into natural ecosystems. The aim of this study was to analyze the response characteristics of nirS-type denitrifier Paracoccus denitrificans under FF stress and further mine antibiotic-responsive factors in aquatic environment. Phenotypic analysis revealed that FF delayed the nitrate removal with a maximum inhibition value of 82.4% at exponential growth phase, leading to nitrite accumulation reached to 21.9-fold and biofilm biomass decreased by ~38.6%, which were due to the lower bacterial population count (P < 0.01). RNA-seq transcriptome analyses indicated that FF treatment decreased the expression of nirS, norB, nosD and nosZ genes that encoded enzymes required for NO2- to N2 conversion from 1.02- to 2.21-fold (P < 0.001). Furthermore, gene associated with the flagellar system FlgL was also down-regulated by 1.03-fold (P < 0.001). Moreover, 10 confirmed sRNAs were significantly induced, which regulated a wide range of metabolic pathways and protein expression. Interestingly, different bacteria contained the same sRNAs means that sRNAs can spread between them. Overall, this study suggests that the denitrification of nirS-type denitrifiers can be hampered widely by FF and the key sRNAs have great potential to be antibiotic-responsive factors.

Antibiotic-induced role interchange between rare and predominant bacteria retained the function of a bacterial community for denitrifying quinoline degradation.

AIM: Quinoline is a recalcitrant pollutant in coking wastewater which has been broadly investigated with many isolates possessing aerobic quinoline-degrading ability. However, studies on anaerobic degradation and the corresponding bacteria are very scarce. This study attempted to investigate the role of diverse functional members and the redundancy of quinoline degradation in a lab-scale quinoline denitrifying bioreactor. METHODS AND RESULTS: Antibiotics were added to the batch culture under denitrifying conditions to disturb the microbial community of the quinoline-degrading bioreactor. According to the results, the nitrate removal rate remained stable, and the quinoline removal rate increased by 9·7% after treatment with streptomycin. However, PCoA analysis of 16S rRNA gene sequencing data of these samples indicated a significant shift in microbial community structures. Specifically, 12 operational taxonomic units (OTUs), including OTU1 (Pseudomonas) and OTU2 (Achromobacter), were significantly enriched. OTU1 replaced OTU8 (Thauera) as the most predominant denitrifying quinoline-degrading member. However, OTU8 and other predominant OTUs (Comamonas and Pseudoxanthomonas), which were hypothesized to contribute essentially to quinoline degradation in the origin bioreactor, became almost undetectable. CONCLUSION: Functional redundancy due to high biological diversity allowed the role reversal of predominant quinoline-degrading bacteria and other rare bacteria when disturbed by antibiotic stress. Although the abundance of OTU1 was much lower initially, it replaced the essential role of the predominant member OTU8 in the bioreactor community for quinoline degradation once the environmental condition changed. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicated that the high biological diversity in a wastewater treatment bacterial community is crucial for maintaining the degrading function of organic pollutants, especially in a changing environment due to external disturbance or stress.

New application of rutin: Repair the toxicity of emerging perfluoroalkyl substance to Pseudomonas stutzeri.

Per- and polyfluoroalkyl substances (PFASs) are toxic to microorganisms, thereby affecting microbial communities in sludge and soil, but how to repair the toxicity of microorganisms remains unclear. In this study, rutin, an antioxidant, was added into a culture medium with an aerobic denitrification bacteria, Pseudomonas stutzeri, under the exposure of sodium perfluorononyloxy-benzenesulfonate (OBS) to evaluate the repair mechanisms of rutin to the toxicity of OBS to the bacteria. The results showed that rutin could repair the damage of OBS to cell structures, and reduce the death rates of the bacteria under OBS exposure. The dosage of rutin reduced the effect on the inhibition of denitrification ability of P. stutzeri under OBS exposure. Compared with the bacteria exposed to single OBS, the dosage of rutin resulted in that the death rates recovered from 96.2% to 66.4%, the growth inhibition rate decreased from 46.5% to 15.8%, the total nitrogen removal rate recovered from 66.9% to 100%, and the NO2- content recovered from 34.5 mg/L to 0.22 mg/L. The expressions of key denitrification genes (napA, nirS, norB, nosZ) were recovered after adding rutin under OBS exposure. Rutin changed the positive rate of reactive oxygen species, the relative superoxide dismutase and catalase activities in the bacteria which exposed to OBS. The mechanism by which rutin repaired the toxicity of OBS to P. stutzeri related to inhibiting the activities of antioxidant and denitrification enzymes rather than affecting the expressions of genes involved in these enzymes. This study sheds light on the repair method of micro-organics and reveals the repair mechanisms under PFASs exposure.

Effects of carbon nanotube on denitrification performance of Alcaligenes sp. TB: Promotion of electron generation, transportation and consumption.

Multi-walled carbon nanotubes (MWCNTs) promote biodegradation in water treatment, but the effect of MWCNT on denitrification under aerobic conditions is still unclear. This investigation focused on the denitrification performance of MWCNT and its toxic effects on Alcaligenes sp. TB which showed that 30 mg/L MWCNTs increased NO3- removal efficiency from 84% to 100% and decreased the NO2-and N2O accumulation rates by 36% and 17.5%, respectively. Nitrite reductase and nitrous oxide reductase activities were further increased by 19.5% and 7.5%, respectively. The mechanism demonstrated that electron generation (NADH yield) and electron transportation system activity increased by 14.5% and 104%, respectively. Cell membrane analysis found that MWCNT caused an increase in polyunsaturated fatty acids, which had positive effects on electron transportation and membrane fluidity at a low concentration of 96 mg/kg but caused membrane lipid peroxidation and impaired membrane integrity at a high concentration of 115 mg/L. These findings confirmed that MWCNT affects the activity of Alcaligenes sp. TB and consequently enhances denitrification performance.

Impact of phenol on the performance, kinetics, microbial communities and functional genes of an autotrophic denitrification system.

Nitrate and phenol often co-occur in wastewater because of the complex industrial and agricultural processes, while the impacts of phenol on autotrophic denitrification remain unclear. Here, a sulfur and hydrogen-oxidizing autotrophic denitrification reactor was established, and the effects of different concentrations of phenol on the nitrate removal performance, kinetics, microbial communities, and functional genes were investigated. Increasing concentrations of phenol significantly decreased the denitrification efficiency in the reactor. The kinetic data indicate the limitation of nitrate diffusion may be one of reasons. Increasing phenol concentrations declined the activities of nitrate and nitrite reductases and induced the production of reactive oxygen species (ROS) and the release of lactate dehydrogenase (LDH), suggesting potential toxicity to the denitrifying consortium. Denitrifying gene nirK was most sensitive to phenol stresses in the reactor. In addition, Thauera was the predominant genus in system with and without phenol, Bacillus was enriched under high phenol concentrations.

The therapeutic value of protein (de)nitrosylation in experimental septic shock.

Cardiovascular dysfunction and organ damage are hallmarks of sepsis and septic shock. Protein S-nitrosylation by nitric oxide has been described as an important modifier of protein function. We studied whether protein nitrosylation/denitrosylation would impact positively in hemodynamic parameters of septic rats. Polymicrobial sepsis was induced by cecal ligation and puncture. Female Wistar rats were treated with increasing doses of DTNB [5,5'-dithio-bis-(2-nitrobenzoic acid)] 30min before or 4 or 12h after sepsis induction. Twenty-four hours after surgery the following data was obtained: aorta response to phenylephrine, mean arterial pressure, vascular reactivity to phenylephrine, biochemical markers of organ damage, survival and aorta protein nitrosylation profile. Sepsis substantially decreases blood pressure and the response of aorta rings and of blood pressure to phenylephrine, as well as increased plasma levels of organ damage markers, mortality of 60% and S-nitrosylation of aorta proteins increased during sepsis. Treatment with DTNB 12h after septic shock induction reversed the loss of response of aorta rings and blood pressure to vasoconstrictors, reduced organ damage and protein nitrosylation and increased survival to 80%. Increases in protein S-nitrosylation are related to cardiovascular dysfunction and multiple organ injury during sepsis. Treatment of rats with DTNB reduced the excessive protein S-nitrosylation, including that in calcium-dependent potassium channels (BKCa), reversed the cardiovascular dysfunction, improved markers of organ dysfunction and glycemic profile and substantially reduced mortality. Since all these beneficial consequences were attained even if DTNB was administered after septic shock onset, protein (de)nitrosylation may be a suitable target for sepsis treatment.

Comprehensive analyses of functional bacteria and genes in a denitrifying EGSB reactor under Cd(II) stress.

A bench-scale expanded granular sludge bioreactor (EGSB) was continuously operated to treat synthesized high-nitrate industrial wastewater with increasing bivalent cadmium (Cd(II)) stress. The bioreactor showed nearly complete nitrate removal regardless of Cd(II) loadings, while nitrite accumulated in the effluent when influent Cd(II) loading was over 64 mg/L. Mi-seq sequencing of 16S rRNA gene amplicons elucidated that denitrifiers had decreasing abundances while biodiversity showed increasing trend as the Cd(II) loading increased. In the bioreactor, genera Halomonas, Thauera, Pseudomonas, and Zoogloea played major roles in the denitrification under lower Cd(II) loadings (< 32 mg/L), while Halomonas sp. KM-1 and Halomonas sp. BC04 acted as the crucial Cd-resistant denitrifiers under 128 mg/L Cd(II) loading. Metagenomic analyses and real-time quantitative PCR consistently indicated that napA encoding nitrate reductase was the predominant denitrifying gene, that could be mainly functioning on the efficient nitrate removal. Statistical analyses revealed the significantly positive correlation between Halomonas and nirS gene, both of which were functionally responsible for nitrite reduction. The obtained results may be practically useful for regulation and optimization of the biological processes to treat industrial wastewater containing high levels of nitrate and Cd(II).

Simultaneous nitrification and denitrification with different mixed nitrogen loads by a hypothermia aerobic bacterium.

Microorganism with simultaneous nitrification and denitrification ability plays a significant role in nitrogen removal process, especially in the eutrophic waters with excessive nitrogen loads. The nitrogen removal capacity of microorganism may suffer from low temperature or nitrite nitrogen source. In this study, a hypothermia aerobic nitrite-denitrifying bacterium, Pseudomonas tolaasii strain Y-11, was selected to determine the simultaneous nitrification and denitrification ability with mixed nitrogen source at 15 °C. The sole nitrogen removal efficiencies of strain Y-11 in simulated wastewater were obtained. After 24 h of incubation at 15 °C, the ammonium nitrogen fell below the detection limit from an initial value of 10.99 mg/L. Approximately 88.0 ± 0.33% of nitrate nitrogen was removed with the initial concentration of 11.78 mg/L and the nitrite nitrogen was not detected with the initial concentration of 10.75 mg/L after 48 h of incubation at 15 °C. Additionally, the simultaneous nitrification and denitrification nitrogen removal ability of P. tolaasii strain Y-11 was evaluated using low concentration of mixed NH4+-N and NO3--N/NO2--N (about 5 mg/L-N each) and high concentration of mixed NH4+-N and NO3--N/NO2--N (about 100 mg/L-N each). There was no nitrite nitrogen accumulation at the time of evaluation. The results demonstrated that P. tolaasii strain Y-11 had higher simultaneous nitrification and denitrification capacity with low concentration of mixed inorganic nitrogen sources and may be applied in low temperature wastewater treatment.

Roxarsone exposure jeopardizes nitrogen removal and regulates bacterial community in biological sequential batch reactors.

Roxarsone is widely present in wastewaters of many animal farms in China. However, little is known about how long-term roxarsone exposure influences the nitrogen removal of biological wastewater treatment in agricultural settings. Here we investigated the nitrogen removal performance of a biological sequential batch reactor (SBR) and the changes of bacterial community, upon long-term roxarsone exposure. The long-term roxarsone dosing decreased the SBR nitrogen removal by 52.4%, with an immediate inhibition on denitrification and a delayed inhibition on nitrification. The analyses of bacterial enzymatic activities and 16 S rRNA sequencing revealed that bacterial activities generally decreased, and the nitrogen-cycling bacterial community was changed, particularly by the decrease (Acinetobacter and Methylophilaceae), persistence (Flavobacterium and Methylotenera), and emergence (Aeromonas) of certain bacterial genera. Overall, chronic roxarsone exposure could suppress nitrification and denitrification, which may even have broad implications on the use efficiency and cycling of nitrogen in agroecosystems.

Effect of the nitrification inhibitor (3, 4-dimethylpyrazole phosphate) on the activities and abundances of ammonia-oxidizers and denitrifiers in a phenanthrene polluted and waterlogged soil.

Through a 60-day microcosm incubation, the effect of 3, 4-dimethylpyrazole phosphate (DMPP) on the activities and abundances of ammonia-oxidizers and denitrifiers in phenanthrene-polluted soil was investigated. Five treatments were conducted for clean soil (CK), phenanthrene added (P), phenanthrene and DMPP added (PD), phenanthrene and urea added (PU), and phenanthrene, urea, and DMPP added (PUD) soils. The results indicate that the potential nitrification rate (PNR) in the P treatment was significantly higher than that in the PD treatment only on day 7, whereas the PNR in the PU treatment was significantly higher than that in the PUD treatment on each sampling day. The abundance of soil ammonia-oxidizing bacteria (AOB) in the PU treatment was significantly higher than that in the PUD treatment on each sampling day. Moreover, the abundance of AOB but rather than the ammonia-oxidizing archaea (AOA) had significantly positive correlation with soil PNR (P < 0.05). DMPP showed no obvious effect on the soil denitrification enzyme activity (DEA), which could have inhibited the abundances of denitrification-related narG, nirS, and nirK genes. The results of this study should provide a deeper understanding of the interaction between soil polycyclic aromatic hydrocarbons (PAH) contamination, ammonia oxidization, and denitrification, and offer valuable information for assessing the potential contribution of denitrification for soil PAH elimination.

Acute Impact of Chlortetracycline on Nitrifying and Denitrifying Processes.

In the current study, sequential nitrification and anoxic experiments in synthetic municipal wastewater were exposed to 0.5 to 100 mg/L of chlortetracycline for 24 h to evaluate acute impact on the nitrification, and denitrification processes of biological treatment. Both processes were significantly (p < 0.05) inhibited at >50 mg/L of chlortetracycline, and the results revealed that nitrification was adversely affected by chlortetracycline compared with the anoxic process. In nitrification, chemical oxygen removal (COD) and ammonia oxidation kinetics were 50% inhibited at 10 mg chlortetracycline/L, and nitrite oxidation kinetics at 0.5 mg chlortetracycline/L. Likewise, in the anoxic process, 14 and 10 mg/L of chlortetracycline inhibited 50% of COD removal and nitrate reduction kinetics, respectively. In nitrification and denitrification, 90% of chlortetracycline was removed by adsorbing onto sludge suspended solids. In addition, a higher chlortetracycline concentration in anoxic effluent, compared with aerobic effluents, indicated a dissimilarity in the composition of sludge solids, pH, and biomass production for both processes.

Effects of residual organics in municipal wastewater on hydrogenotrophic denitrifying microbial communities.

Hydrogenotrophic denitrification is promising for tertiary nitrogen removal from municipal wastewater. To reveal the influence of residual organics in municipal wastewater on hydrogenotrophic denitrifiers, we adopted high-throughput 16S rRNA gene amplicon sequencing to examine microbial communities in hydrogenotrophic denitrification enrichments. Using effluent from a municipal wastewater treatment plant as water source, COD, nitrate and pH were controlled the same except for a gradient of biodegradable carbon (i.e., primary effluent (PE), secondary effluent (SE), or combined primary and secondary effluent (CE)). Inorganic synthetic water (IW) was used as a control. Hydrogenophaga, a major facultative autotroph, accounted for 17.1%, 5.3%, 32.7% and 12.9% of the sequences in PE, CE, SE and IW, respectively, implicating that Hydrogenophaga grew well with or without organics. Thauera, which contains likely obligate autotrophic denitrifiers, appeared to be the most dominant genera (23.6%) in IW and accounted for 2.5%, 4.6% and 8.9% in PE, CE and SE, respectively. Thermomonas, which is related to heterotrophic denitrification, accounted for 4.2% and 7.9% in PE and CE fed with a higher content of labile organics, respectively. In contrast, Thermomonas was not detected in IW and accounted for only 0.6% in SE. Our results suggest that Thermomonas are more competitive than Thauera in hydrogenotrophic denitrification with biodegradable organics. Moreover, facultative autotrophic denitrifiers, Hydrogenophaga, are accommodating to residual organic in effluent wastewater, thus we propose that hydrogenotrophic denitrification is amenable for tertiary nitrogen removal.

Nitrifier-induced denitrification is an important source of soil nitrous oxide and can be inhibited by a nitrification inhibitor 3,4-dimethylpyrazole phosphate.

Soil ecosystem represents the largest contributor to global nitrous oxide (N2 O) production, which is regulated by a wide variety of microbial communities in multiple biological pathways. A mechanistic understanding of these N2 O production biological pathways in complex soil environment is essential for improving model performance and developing innovative mitigation strategies. Here, combined approaches of the 15 N-18 O labelling technique, transcriptome analysis, and Illumina MiSeq sequencing were used to identify the relative contributions of four N2 O pathways including nitrification, nitrifier-induced denitrification (nitrifier denitrification and nitrification-coupled denitrification) and heterotrophic denitrification in six soils (alkaline vs. acid soils). In alkaline soils, nitrification and nitrifier-induced denitrification were the dominant pathways of N2 O production, and application of the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) significantly reduced the N2 O production from these pathways; this is probably due to the observed reduction in the expression of the amoA gene in ammonia-oxidizing bacteria (AOB) in the DMPP-amended treatments. In acid soils, however, heterotrophic denitrification was the main source for N2 O production, and was not impacted by the application of DMPP. Our results provide robust evidence that the nitrification inhibitor DMPP can inhibit the N2 O production from nitrifier-induced denitrification, a potential significant source of N2 O production in agricultural soils.

Discovery of Fungal Denitrification Inhibitors by Targeting Copper Nitrite Reductase from Fusarium oxysporum.

The efficient application of nitrogenous fertilizers is urgently required, as their excessive and inefficient use is causing substantial economic loss and environmental pollution. A significant amount of applied nitrogen in agricultural soils is lost as nitrous oxide (N2O) in the environment due to the microbial denitrification process. The widely distributed fungus Fusarium oxysporum is a major denitrifier in agricultural soils and its denitrification activity could be targeted to reduce nitrogen loss in the form of N2O from agricultural soils. Here, we report the discovery of first small molecule inhibitors of copper nitrite reductase (NirK) from F. oxysporum, which is a key enzyme in the fungal denitrification process. The inhibitors were discovered by a hierarchical in silico screening approach consisting of pharmacophore modeling and molecular docking. In vitro evaluation of F. oxysporum NirK activity revealed several pyrimidone and triazinone based compounds with potency in the low micromolar range. Some of these compounds suppressed the fungal denitrification in vivo as well. The compounds reported here could be used as starting points for the development of nitrogenous fertilizer supplements and coatings as a means to prevent nitrogen loss by targeting fungal denitrification.

Effects of heavy metals on aerobic denitrification by strain Pseudomonas stutzeri PCN-1.

Effects of heavy metals on aerobic denitrification have been poorly understood compared with their impacts on anaerobic denitrification. This paper presented effects of four heavy metals (Cd(II), Cu(II), Ni(II), and Zn(II)) on aerobic denitrification by a novel aerobic denitrifying strain Pseudomonas stutzeri PCN-1. Results indicated that aerobic denitrifying activity decreased with increasing heavy metal concentrations due to their corresponding inhibition on the denitrifying gene expression characterized by a time lapse between the expression of the nosZ gene and that of the cnorB gene by PCN-1, which led to lower nitrate removal rate (1.67∼6.67 mg L-1 h-1), higher nitrite accumulation (47.3∼99.8 mg L-1), and higher N2O emission ratios (5∼283 mg L-1/mg L-1). Specially, promotion of the nosZ gene expression by increasing Cu(II) concentrations (0∼0.05 mg L-1) was found, and the absence of Cu resulted in massive N2O emission due to poor synthesis of N2O reductase. The inhibition effect for both aerobic denitrifying activity and denitrifying gene expression was as follows from strongest to least: Cd(II) (0.5∼2.5 mg L-1) > Cu(II) (0.5∼5 mg L-1) > Ni(II) (2∼10 mg L-1) > Zn(II) (25∼50 mg L-1). Furthermore, sensitivity of denitrifying gene to heavy metals was similar in order of nosZ > nirS ≈ cnorB > napA. This study is of significance in understanding the potential application of aerobic denitrifying bacteria in practical wastewater treatment.

Low temperature, autotrophic microbial denitrification using thiosulfate or thiocyanate as electron donor.

Wastewaters generated during mining and processing of metal sulfide ores are often acidic (pH < 3) and can contain significant concentrations of nitrate, nitrite, and ammonium from nitrogen based explosives. In addition, wastewaters from sulfide ore treatment plants and tailings ponds typically contain large amounts of inorganic sulfur compounds, such as thiosulfate and tetrathionate. Release of these wastewaters can lead to environmental acidification as well as an increase in nutrients (eutrophication) and compounds that are potentially toxic to humans and animals. Waters from cyanidation plants for gold extraction will often conjointly include toxic, sulfur containing thiocyanate. More stringent regulatory limits on the release of mining wastes containing compounds such as inorganic sulfur compounds, nitrate, and thiocyanate, along the need to increase production from sulfide mineral mining calls for low cost techniques to remove these pollutants under ambient temperatures (approximately 8 °C). In this study, we used both aerobic and anaerobic continuous cultures to successfully couple inorganic sulfur compound (i.e. thiosulfate and thiocyanate) oxidation for the removal of nitrogenous compounds under neutral to acidic pH at the low temperatures typical for boreal climates. Furthermore, the development of the respective microbial communities was identified over time by DNA sequencing, and found to contain a consortium including populations aligning within Flavobacterium, Thiobacillus, and Comamonadaceae lineages. This is the first study to remediate mining waste waters by coupling autotrophic thiocyanate oxidation to nitrate reduction at low temperatures and acidic pH by means of an identified microbial community.

Biomass aggregation influences NaN3 short-term effects on anammox bacteria activity.

The main bottleneck to maintain the long-term stability of the partial nitritation-anammox processes, especially those operated at low temperatures and nitrogen concentrations, is the undesirable development of nitrite oxidizing bacteria (NOB). When this occurs, the punctual addition of compounds with the capacity to specifically inhibit NOB without affecting the process efficiency might be of interest. Sodium azide (NaN3) is an already known NOB inhibitor which at low concentrations does not significantly affect the ammonia oxidizing bacteria (AOB) activity. However, studies about its influence on anammox bacteria are unavailable. For this reason, the objective of the present study was to evaluate the effect of NaN3 on the anammox activity. Three different types of anammox biomass were used: granular biomass comprising AOB and anammox bacteria (G1), anammox enriched granules (G2) and previous anammox granules disaggregated (F1). No inhibitory effect of NaN3 was measured on G1 sludge. However, the anammox activity decreased in the case of G2 and F1. Granular biomass activity was less affected (IC50 90 mg/L, G2) than flocculent one (IC50 5 mg/L, F1). Summing up, not only does the granular structure protect the anammox bacteria from the NaN3 inhibitory effect, but also the AOB act as a barrier decreasing the inhibition.

Determining Multiple Responses of Pseudomonas aeruginosa PAO1 to an Antimicrobial Agent, Free Nitrous Acid.

Free nitrous acid (FNA) has recently been demonstrated as an antimicrobial agent on a range of micro-organisms, especially in wastewater-treatment systems. However, the antimicrobial mechanism of FNA is largely unknown. Here, we report that the antimicrobial effects of FNA are multitargeted. The response of a model denitrifier, Pseudomnas aeruginosa PAO1 (PAO1), common in wastewater treatment, was investigated in the absence and presence of inhibitory level of FNA (0.1 mg N/L) under anaerobic denitrifying conditions. This was achieved through coupling gene expression analysis, by RNA sequencing, and with a suite of physiological analyses. Various transcripts exhibited significant changes in abundance in the presence of FNA. Respiration was likely inhibited because denitrification activity was severely depleted, and decreased transcript levels of most denitrification genes occurred. As a consequence, the tricarboxylic acid (TCA) cycle was inhibited due to the lowered cellular redox state in the FNA-exposed cultures. Meanwhile, during FNA exposure, PAO1 rerouted its carbon metabolic pathway from the TCA cycle to pyruvate fermentation with acetate as the end product as a possible survival mechanism. Additionally, protein synthesis was significantly decreased, and ribosome preservation was evident. These findings improve our understanding of PAO1 in response to FNA and contribute toward the potential application for use of FNA as an antimicrobial agent.